The biochemical modification of human neutrophil (PMN) tubuling microtubules was studied by monitoring the post-translational incorporation of tyrosine into tubulin Alpha-chains. The data indicate that a variety of stimuli, such as the peptide chemoattractant fmet-leu-phe, the Ca2+ ionophore A23187 and phorbol myristate acetate (PMA), cause a 2-3 fold stimulation of PMN tubulin tyrosinolation that is closely associated with the PMN redox state. The above stimuli fail to induce stimulation in normal PMN under reduced and anaerobic conditions and also in PMN from patients with chronic granulomatous disease, which are deficient in oxidative metabolism. Extracellular Ca2+ and the cellular Ca2+ regulatory protein, calmodulin, are necessary for the modulation of PMN tubulin tyrosinolation, although there are significant differences in the CA2+ sensitivity of various stimuli. In studies to localize the reaction at a subcellular level, tightly associated tubulin has been detected in isolated PMN fractions enriched in plasma membranes, azurophil granules and specific granules, that could be tyrosinolated in vitro in the presence of exogenous ligase, the enzyme that catalyzes the reaction. Endogenous ligase was detected in PMN azurphil granules. This is the first demonstration of an intracellular organelle-associated ligase in its functional form. Further studies utilizing a monoclonal antibody, YL 1/2 which recognizes only the tyrosinolated form of Alpha-tubulin, reveal a preferential stimulation of tyrosinolation in plasma membrane-associated tubulin of fmet-leu-phe-stimulated PMN. A significant stimulation was also observed in the cytoplasmic tubulin fraction. Unlike in intact PMN, organelle-depleted PMN cytoplasts do not respond to fmet-leu-phe, although they have a significant basal level of tyrosinolation. These results provide further insights into the mechanism of modulation of tubulin tyrosinolation in PMN and demonstrate the sub-cellular localization of the reaction in resting and stimulated PMN.